Drug Efficacy Evaluation, ADME/T Analysis, And Crystal Engineering In Drug Discovery

Drug discovery is the initial phase of the drug life cycle. Modern drug discovery processes based on active candidate selection usually commences with the identification of an appropriate target with the understanding of its role in the disease process, followed by a screening step in search for candidate lead compounds that can interact with the target, after which rigorous investigation and optimization are taken over selected compounds, to test their pharmacodynamics, pharmacokinetics, safety, toxicity, dosage, and efficacy. Only lead compounds which manifest satisfactory efficacy and meet requisite safety standards can be selected as candidate drug, and subsequently be thoroughly investigated, optimized, and prepared for human test in the pre-clinical phase.The demand of reliable pharmaceutical screening models, which is indispensable in the first step of drug discovery, calls for new advances in biological sciences and state-of-the-art biotechnologies. Besides, an array of problems and challenges may be encountered during the investigation and the optimization phase of the lead compound, e.g., undetermined drug target, obscured dose effect relationship, undesired pharmacokinetic properties, potential toxicity, etc. To address these aforementioned issues, novel and untraditional techniques and approaches may need to be designed and developed.This manuscript presents a number of pieces of research concerning application of new models and novel methods in the drug discovery processes, employing pinocembrin and nitrendipine as specific examples.PartⅠMechanism study of the neuroprotective effect of PNCB on ischemia/reperfusion ratsThe neuro-protective effect of PNCB on ischemia-reperfusion was previously studied. Evidence showed that when the administrated dose goes above a certain level, the neuro-protective effect of PNCB would be lessened rather than strengthened. This phenomenon is probably due to the activation of dissimilar targets and signal transduction pathways under differing dosage. In this work, possible mechanisms to interpret the neuro-protective effect of PNCB will be discussed, by leveraging the four-vessel occlusion model.The administration of 5mg/kg PNCB 30min after ischemia-reperfusion achieved the optimal effect in this study, which could be attributed to series of signal transduction pathways that lead to CREB activation, and the possible contribution from its upstream factors including TORC-1/CREB, CaMKII, PI3K/Akt. p38MAPK, and JNK pathways may also contributed to the neuro-protective effects of PNCB. These factors were effective at different stages after ischemia reperfusion and PNCB administration, and responded differently to certain dose of PNCB. As TORC-1/CREB, CaMKII and PI3K/Akt were downstram targets of NMDA receptors, the neuro-protective effects of PNCB could be attained by the inhibition of non-synaptic NMDA receptors. Extremely high concentration of PNCB produces sub-optimal protection because if could trigger negative feedbacks suppressing the protective signal transduction pathways and depreciate the neuro-protective effects via the inhibition of synaptic NMDA receptors.PartⅡIn this section, Aplysia californica was used to assess the possible effects of PNCB.Neural electrophysiological effects of PNCB on Aplysia sensory/motor neuron co-cultures were evaluated. Results showed that PNCB incubation reversibly reduced the EPSP amplitude of Aplysia SN/L7 co-cultures. Linear relationships between PNCB concentration and EPSP amplitude could be established when PNCB concentration was within 10～100μM and 100～400μM, respectively, being in opposite directions. Also observed was that the incubation with 10μM PNCB reversibly inhibited 5-HT induced synaptic facilitation. However, no effect was detected when 5-HT was applied after the removal of PNCB. Possible interpretation could be relevant to the complicated effects of PNCB towards pre-and post-synaptic ion channels.New protein synthesis was required in synaptic facilitation. rRNA transcription was boosted in the early phase. By marking nucleolar protein fibrillarin, DFC region of nucleoli was visualized, and Aplysia nucleolar morphology was related with synaptic facilitation. Results were confirmed by using excitatory neurotransmitter serotonin to induce synaptic facilitation, and by using PKC inhibitor chelyrethrine, PARP-1 inhibitor PJ34 to induce synaptic inhibition. Based on the calculation, the effect of PNCB toward the nucleolar morphology was studied quantitatively, from which we concluded that 10μM PNCB could activate the rRNA transcription machine of sensory neuron while 40μM PNCB gave rise to an inhibitory effect.By using real-time PCR techinique, differentiate expression of aging related genes in Aplysia neurons were investigated. Administration of PNCB can lower K+channel, GluR5 and fibrillarin expressions in R2, but enhanced APP, fibrillarin expressions in LP11.All the results indicate that PNCB yields a spectrum of pharmacological effects towards Aplysia. This discovery will help explain the mechanism of neuro-pharmacological effects of PNCB in other animals as well.PartⅢPNCB is quickly metabolized after administration, and the recovery level of PNCB is quite low in excreta. To gain an insight into the final fate of the drug-related materials concerning drug safety, mass balance study of PNCB is required.In this study, a mathematical method was developed to facilitate quantitative analysis of metabolites and mass balance study of PNCB. Linear relationships of high performance liquid chromatography (HPLC) areas of PNCB and those of PNCB metabolites were established based on in vitro hepatic subfractional incubations. The reliability of the mathematical model was tested by leave-one-out cross validation (LOOCV), with results suggesting that the proposed model was able to achieve quite strong correspondence. When the in vitro developed equations were applied in the mass balance study in rats, it was found that 48h after pinocembrin administration, drug cumulative excretion ratio of most rats were close to 100%.As an important part of pre-clinical safety evaluation, a 29-day-toxicokinetics study was carried out in beagle dogs. Administration dose of PNCB was 5mg/kg,20mg/kg, and 80mg/kg, respectively. Upon repeated administration, systemic exposure to PNCB was increased, especially at 20mg/kg/day. The highest accumulation was observed at 80mg/kg/day in liver and kidney. Results indicate that potential toxicity of PNCB should be closely monitored in the clinical trial, especial toxicity to the liver and kidney.Human plasma protein binding rate is a critical parameter in the study of drug disposition, drug efficacy and drug-drug interactions. Using a novel method of micro-dialysis, human plasma protein binding rate of PNCB was estimated to be 96.9%。Part IVThe purpose of this study is to improve the solubility, bioavailability and efficacy of nitrendipine by crystal engineering. A new crystal form of nitrendipine, Form IV, was prepared. To compare NTD-IV to form NTD-I, powder X-ray diffraction, differential scanning calorimetry and scanning electron microscopy were employed. In vitro dynamic solubility test and permeation test were carried out; bioavailability and blood-pressure-lowering effect were also investigated in rats.Results showed that NTD-IV was a novel crystal form of nitrendipine. Compared to NTD-I, the initial dissolution rate of NTD-IV was 133% faster, and the permeated amount of NTD-IV through Caco-2 cell monolayer was 2 times higher than that of NTD-I during 2h study. The relative bioavailability of NTD-IV in rats was 292% of form I. When 18mg/kg nitrendipine was orally administrated to rats, NTD-IV could significantly lower both diastolic and systolic pressures, while the administration of form I showed little difference from normal saline.Bioavailability of novel tablets of nitrendipine, while using NTD-IV as the raw material, was investigated in beagle dogs. Compared to the commercially available tablets, the relative bioavailability of the new formulation was 134.6%.This study indicates that the solubility, bioavailability and effecacy of nitrendipine were enhanced by appropriate crystal engineering. The novel nitrendipine tablets could be more potent to treat hypertension than commercially available tablets in China.Aforementioned studies investigated druggability of PNCB and NTD-IV by evaluating drug efficacy, safety, material form etc. Results showed that the combination of evaluation methods can help understand characters of the chemical compounds, improving the efficiency and lowering the cost of drug development.